Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by
            <i>Corynebacterium glutamicum</i>

Becker, Jörg; Klopprogge, Corinna; Schröder, Hartwig; Wittmann, Christoph

doi:10.1128/aem.01942-09

Cited by 107 publications

(59 citation statements)

References 21 publications

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“…Malic enzyme (ME; EC 1.1.1.40) activity was determined as described previously (38). As a positive control, an isocitrate dehydrogenase assay was used (39). Enzyme activities were calculated from the change in the absorbance.…”

Section: Methodsmentioning

confidence: 99%

The Key to Acetate: Metabolic Fluxes of Acetic Acid Bacteria under Cocoa Pulp Fermentation-Simulating Conditions

Adler

Frey

Berger

et al. 2014

Appl Environ Microbiol

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c Acetic acid bacteria (AAB) play an important role during cocoa fermentation, as their main product, acetate, is a major driver for the development of the desired cocoa flavors. Here, we investigated the specialized metabolism of these bacteria under cocoa pulp fermentation-simulating conditions. A carefully designed combination of parallel 13 C isotope labeling experiments allowed the elucidation of intracellular fluxes in the complex environment of cocoa pulp, when lactate and ethanol were included as primary substrates among undefined ingredients. We demonstrate that AAB exhibit a functionally separated metabolism during coconsumption of two-carbon and three-carbon substrates. Acetate is almost exclusively derived from ethanol, while lactate serves for the formation of acetoin and biomass building blocks. Although this is suboptimal for cellular energetics, this allows maximized growth and conversion rates. The functional separation results from a lack of phosphoenolpyruvate carboxykinase and malic enzymes, typically present in bacteria to interconnect metabolism. In fact, gluconeogenesis is driven by pyruvate phosphate dikinase. Consequently, a balanced ratio of lactate and ethanol is important for the optimum performance of AAB. As lactate and ethanol are individually supplied by lactic acid bacteria and yeasts during the initial phase of cocoa fermentation, respectively, this underlines the importance of a well-balanced microbial consortium for a successful fermentation process. Indeed, AAB performed the best and produced the largest amounts of acetate in mixed culture experiments when lactic acid bacteria and yeasts were both present.A cetic acid bacteria (AAB) play an important role in cocoa fermentation (1). During fermentation of the pulp that surrounds the cocoa beans, they form acetate. Acetate then diffuses into the beans (2-4), where it initiates a cascade of chemical and biochemical reactions leading to precursor molecules for cocoa flavor (2, 5, 6). Potential substrates for AAB are lactate and ethanol, which are individually produced by lactic acid bacteria (LAB; mainly Lactobacillus fermentum) and yeasts (diverse yeasts such as Saccharomyces cerevisiae, Hanseniaspora opuntiae, and Candida krusei), respectively, during the fermentation process (6-12). Hereby, the degradation of lactate by AAB is desired, since the remaining lactate may provide an off flavor in the final cocoa product (11,13,14). In recent years, AAB have been extensively analyzed for their contribution to cocoa fermentation. Obviously, the most prevalent AAB species is Acetobacter pasteurianus (13,(15)(16)(17). In addition, Acetobacter ghanensis and Acetobacter senegalensis are found during spontaneous cocoa bean fermentation (9,13,17,18). Further studies provided first insights into the basic microbiological properties of such strains and macroscopic dynamics during cocoa pulp fermentation (12,15,(18)(19)(20). At this point, it appears to be relevant to resolve the metabolic contribution of AAB in greater detail. The basic know...

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Section: Methodsmentioning

confidence: 99%

The Key to Acetate: Metabolic Fluxes of Acetic Acid Bacteria under Cocoa Pulp Fermentation-Simulating Conditions

Adler

Frey

Berger

et al. 2014

Appl Environ Microbiol

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“…The strains used in the present work comprised the wild-type Corynebacterium glutamicum ATCC 13032 (ATCC, Manassas, VA), the lysine producer C. glutamicum 11424, exhibiting different modifications of the lysine biosynthetic pathway and anaplerotic carboxylation (2), and the diaminopentane producer C. glutamicum DAP-3c, rationally derived from C. glutamicum 11424 by codon-optimized expression of lysine decarboxylase (13). For performing genetic engineering work, Escherichia coli strains DH5␣ and NM522 and plasmids pTc and pClik int sacB were applied as described previously (13).…”

Section: Methodsmentioning

confidence: 99%

“…The activity of diaminopentane acetyltransferase was determined in crude cell extract which was prepared from cells grown in minimal medium, as described above. Preparation of crude cell extract using 50 mM Tris-HCl buffer, pH 7.8, and 0.75 mM dithiothreitol (DTT) as a disruption buffer and determination of protein concentration (6) were performed as described previously (2,13). Assays were performed in a total volume of 1 ml containing 50 mM Tris-HCl buffer (pH 7.8), 0.25 mM acetyl-coenzyme A (CoA), and 200 l cell extract at 30°C.…”

Section: Methodsmentioning

confidence: 99%

Identification and Elimination of the Competing N -Acetyldiaminopentane Pathway for Improved Production of Diaminopentane by Corynebacterium glutamicum

Kind

Jeong

Schröder

et al. 2010

Appl Environ Microbiol

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The present work describes the development of a superior strain of Corynebacterium glutamicum for diaminopentane (cadaverine) production aimed at the identification and deletion of the underlying unknown N-acetyldiaminopentane pathway. This acetylated product variant, recently discovered, is a highly undesired by-product with respect to carbon yield and product purity. Initial studies with C. glutamicum DAP-3c, a previously derived tailor-made diaminopentane producer, showed that up to 20% of the product occurs in the unfavorable acetylated form. The strain revealed enzymatic activity for diaminopentane acetylation, requiring acetyl-coenzyme A (CoA) as a donor. Comparative transcriptome analysis of DAP-3c and its parent strain did not reveal significant differences in the expression levels of 17 potential candidates annotated as N-acetyltransferases. Targeted single deletion of several of the candidate genes showed NCgl1469 to be the responsible enzyme. NCgl1469 was functionally assigned as diaminopentane acetyltransferase. The deletion strain, designated C. glutamicum DAP-4, exhibited a complete lack of N-acetyldiaminopentane accumulation in medium. Hereby, the yield for diaminopentane increased by 11%. The mutant strain allowed the production of diaminopentane as the sole product. The deletion did not cause any negative growth effects, since the specific growth rate and glucose uptake rate remained unchanged. The identification and elimination of the responsible acetyltransferase gene, as presented here, display key contributions of a superior C. glutamicum strain producing diaminopentane as a future building block for bio-based polyamides.Polyamides are polymers containing monomers joined by peptide bonds, with examples being nylons, aramids, and polyaspartates. They are commonly used in textiles, automotives, carpet, and sportswear due to their extreme durability and strength. The most prominent products, polyamides PA 6 and PA 6.6, have an annual market volume of about 6 million tons. Currently, polyamides are derived via chemical routes from fossil raw materials. Due to the shortage of these resources and problems of escalating CO 2 production and global warming linked to the underlying processes, bio-based production using renewable resources arises as a promising alternative (11,23,27). In this context, fermentative production of diaminopentane (cadaverine) as a monomer building block for polyamides has recently come into focus (19). Using diaminopentane derived from microbial biosynthesis, polymerization with appropriate bioblocks, such as succinate (9, 22), provides completely bio-based products. Moreover, polyamides based on diaminopentane reveal excellent material properties (7). The experience of the past clearly shows that a superior production strain with a high yield, level of productivity, and titer requires substantial modification at different key points of the metabolism, which have to be identified by careful investigation of the underlying metabolism (28). The major targets typically compri...

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“…Moreover, other significant effects observed in the mutant E. coli strain included upregulation of oxPPP enzymes and the anaplerotic glyoxylate pathway. Similarly, redirection from the TCA cycle to anaplerosis has also been reported for an IDH knockdown of C. glutamicum [248]. However, with respect to citrate synthase, contradictory results have been obtained.…”

Section: Introductionmentioning

confidence: 73%

“…The exact effects of IDH disruption were dependent on the specific conditions and species used, but some general trends were apparent [247][248][249][250][251]. First, all IDH mutants investigated were glutamate auxotrophs.…”

Section: Introductionmentioning

confidence: 99%

Thermococcus kodakarensis : the key to affordable biohydrogen production

Spaans¹

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Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum

Cited by 107 publications

References 21 publications

The Key to Acetate: Metabolic Fluxes of Acetic Acid Bacteria under Cocoa Pulp Fermentation-Simulating Conditions

The Key to Acetate: Metabolic Fluxes of Acetic Acid Bacteria under Cocoa Pulp Fermentation-Simulating Conditions

Identification and Elimination of the Competing N -Acetyldiaminopentane Pathway for Improved Production of Diaminopentane by Corynebacterium glutamicum

Thermococcus kodakarensis : the key to affordable biohydrogen production

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